Mol. Hum. Reprod. Advance Access originally published online on July 22, 2005
Molecular Human Reproduction 2005 11(7):523-529; doi:10.1093/molehr/gah188
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Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation
1Reproductive Biology and Genetics Group, Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK, 2Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham B15 2TG, UK, 3Centre for Research in Contraceptive and Reproductive Health, Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA, 4Department of Medical Biochemistry and Immunology, Cardiff University, Cardiff, CF14 4XN, UK and 5School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
6 To whom correspondence should be addressed at: Reproductive Biology and Genetics Group, University of Birmingham, Institute of Biomedical Research, Edgbaston, Birmingham, B15 2TT, UK. E-mail: l.lefievre{at}bham.ac.uk
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Abstract |
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Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook® Sydney IVF medium, Cook® Sydney IVF sperm buffer, Earle’s balanced salt medium (capacitating medium) or a modified Earle’s balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37ºC and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.
Key words: acrosome reaction/hyperactivaton/PKA-dependent serine/protein kinase A/threonine phosphorylation/tyrosine phosphorylation
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Introduction |
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After ejaculation, mammalian sperm undergo many biochemical and functional changes that render the cells competent to fertilize an oocyte, a process termed capacitation (de Lamirande et al., 1997
; Visconti et al., 2002). The modifications observed in human spermatozoa include an efflux of cholesterol from the plasma membrane (Cross, 1998; Osheroff et al., 1999), increased activity of adenylyl cyclase, elevation of cyclic adenosine monophosphate (cAMP) and stimulation of protein kinase A (PKA) (Parinaud and Milhet, 1996; Chen et al., 2000; Lefièvre et al., 2002), increased protein serine/threonine and tyrosine phosphorylation (Leclerc et al., 1996; Osheroff et al., 1999; O’Flaherty et al., 2004), changes in [Ca2+]i (Baldi et al., 1991) and events involving components of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPK) (Luconi et al., 1998; de Lamirande and Gagnon, 2002). Functional changes associated with this include alterations in the pattern of motility (termed hyperactivation) and acquisition of the ability to undergo the acrosome reaction in response to physiological or non-physiological agonists (de Lamirande et al., 1997).
Human sperm capacitation can be induced during in vitro incubation. Many well-characterized incubation media have been shown to support capacitation [Tyrodes, Hams-F10, Biggers, Whitten and Whittingham (BWW), supplemented Earle’s balanced salt solution (sEBSS), Hank’s balanced salt solution (HBSS) and Synthetic Human Tubal Fluid (HTF)] (Edwards et al., 1980; Mortimer, 1986; Calvo et al., 1993) as well as supplementation of media with uncharacterized biological fluids [fetal cord serum ultrafiltrate (FCSu), follicular fluid (FF), follicular fluid ultrafiltrate (FFu)] and progesterone (found in the female tract). It is not known whether all these treatments induce capacitation through the same signalling pathways or with the same degree of efficacy. In addition to the wide range of capacitating media used, the conditions used by different laboratories for in vitro capacitation vary in both the sperm concentration used and, in particular, the period for which cells are incubated (ranging from 2 to 24 h). These are important factors to take into account. For example, it has been reported that short co-incubation of gametes (1–2 h) during in vitro fertilization (IVF) significantly improves embryo quality in comparison with longer incubations (20 h) (Quinn et al., 1998; Dirnfeld et al., 1999; Kattera and Chen, 2003). Prolonged co-incubation of gametes appears to produce high levels of reactive oxygen species that are detrimental to sperm motility and viability (de Lamirande and Gagnon, 1995) and induce zona hardening for the oocyte as well as affecting embryo viability (Gianaroli et al., 1996).
The discovery of sperm capacitation, independently by Chang (1951) and Austin (1952), was essential for the development of techniques and media for in vitro preparation of fertilization-competent human spermatozoa (Bavister, 2002). During the development of IVF methodologies, specialized fertilization media have been developed on the basis of their efficacy and reliability in the IVF clinic. IVF medium, which is known to support fertilization leading to pregnancy and live births, is now commercially available, standardized and widely used. Experimental studies on the dynamics of capacitation in an IVF system (induction of sperm capacitation using an IVF media) are thus potentially of great value in identifying the important functional changes required for successful fertilization. Surprisingly, the methods of sperm preparation for IVF have not been exploited to gain a better understanding of the processes leading to sperm capacitation. Capacitation-related markers have not been closely studied in cells prepared using an IVF system and only recently it has been shown that low levels of tyrosine phosphorylation observed on the sperm flagellum (<7%) are correlated with reduced IVF rates (Sakkas et al., 2003).
The aim of this study was to evaluate sperm capacitation induced by IVF medium in comparison with that occurring in spermatozoa incubated in standard Earle’s balanced salt medium (capacitating medium) used in a nonclinical setting. There appears to be a clear relationship between the activation of PKA and tyrosine phosphorylation in human spermatozoa. However, whether upstream activation of PKA is an indispensable condition for tyrosine kinase activation and/or inactivation of tyrosine phosphatases, leading to tyrosine phosphorylation and further downstream events (modification of motility, acrosome reaction), remains to be clearly established. We have therefore specifically investigated the effects of incubation under capacitating conditions on PKA activity and PKA-dependent serine/threonine phosphorylation and their association with protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction.
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Materials
Percoll was purchased from Amersham Biosciences (Chalfont St Giles, Bucks, UK). Sydney IVF sperm buffer and Sydney IVF medium were obtained from Cook® (Eight Mile Plains, Brisbane, Australia). Serine-threonine phosphatase inhibitors calyculin A and okadaic acid were distributed by Cell Signalling Technology and Calbiochem (Merck Biosciences Ltd., Beeston, Nottingham, UK), respectively. Phospho-(serine/threonine) PKA substrate antibody and anti-phosphotyrosine, recombinant 4G10 were obtained from New England Biolabs Ltd (Hitchin, Hertfordshire, UK) and Upstate (Dundee Technology Park, Dundee, UK), respectively. MicrocellTM-HAC chambers were purchased from Conception Technologies (San Diego, CA, USA). The fluorescence mounting medium was purchased from Dako Cytomation Ltd. (Ely, Cambridgeshire, UK). All other chemicals were purchased from Sigma (Poole, Dorset, UK).
Donors
Research donors were recruited at the Assisted Conception Unit, Birmingham Women’s Hospital (Human Fertilisation and Embryology Authority Centre #0119) in accordance with HFEA Code of Practice under local ethical approval (#2003/239). Donors were normozoospermic in accordance with World Health Organisation (1999) guidelines.
Sample preparation
Semen samples were obtained from healthy volunteers after 2–3 days of sexual abstinence. After liquefaction, 1 ml of semen was layered over 1 ml fractions of 45 and 90% Percoll-made isotonic with M medium (1x; 137 mM NaCl, 2.5 mM KCl, 20 mM HEPES, 10 mM glucose). Samples were centrifuged at 700 g for 20 min. Sperm concentration was determined using an improved Neubauer haemocytometer and at least 400 cells were scored (Kvist and Björndahl, 2002).
Sperm incubation
Percoll-washed spermatozoa were resuspended in a capacitating medium (CM) based upon Earle’s balanced salt medium, and supplemented with albumin and bicarbonate [0.3% bovine serum albumin (BSA), 1 mM NaH2PO4, 5.4 mM KCl, 0.8 mM MgSO4·7H20, 5.6 mM glucose, 2.7 mM sodium pyruvate, 25 mM dl-lactic acid, 1.8 mM CaCl2.H2O, 26 mM NaHCO3, 116 mM NaCl], a non-capacitating HEPES-buffered medium (NCM) adapted from the Earle’s balanced salt medium and lacking both albumin and bicarbonate (NCM; 1 mM NaH2PO4, 5.4 mM KCl, 0.8 mM MgSO4·7H20, 5.6 mM glucose, 2.7 mM sodium pyruvate, 25 mM dl-lactic acid, 1.8 mM CaCl2.H2O, 10 mM HEPES, 116 mM NaCl; additional sodium chloride was added to adjust osmolarity), Cook® Sydney IVF sperm buffer (a HEPES-buffered solution containing 10 mM NaHCO3) or Cook® Sydney IVF medium (a bicarbonate buffered medium containing 25 mM NaHCO3). Concentration of spermatozoa was diluted to 6 106 cells/ml and cells were incubated for various time periods at 37ºC, 5% CO2. Incubation in NCM and IVF sperm buffer were performed at 37ºC in air tight vials to prevent pH changes because of the equilibration of bicarbonate. All T0 samples were taken immediately following re-suspension into medium (a maximum of 1 min incubation).
Evaluation of PKA activity
Spermatozoa incubated in NCM, CM or IVF medium were snap frozen in liquid nitrogen at T0, 5, 15, 30 and 90 min. PKA activity was measured as previously described (Lefièvre et al., 2002) using Kemptide as a substrate. Briefly, frozen sperm samples (triplicates) were supplemented with 2x assay cocktail (final concentrations: 100 mM Kemptide, 1 µCi (
-32P) ATP, 40 mM ATP, 1% (v/v) Triton X-100, 1 mg/ml BSA, 10 mM MgCl2, 40 mM 
-glycerophosphate, 5 mM p-nitrophenyl phosphate, 25 mM HEPES (pH 7.4), EDTA-free complete protease inhibitor cocktail and 100 µM vanadate). Another triplicate of the same sperm sample was measured in conditions that maximize PKA activity (addition of 1 mM dbcAMP + 100 µM 3-isobutyl-1-methylxanthine (IBMX) to the assay mixture) to eliminate the possibility that differences in PKA activity observed would be because of the different amounts of enzyme or spermatozoa in the PKA assays. Both sets of triplicates were then incubated for 15 min at 37°C, and the reaction was stopped by the addition of trichloroacetic acid (10% final concentration). Samples were cooled and centrifuged at room temperature for 3 min at 10 000 g. The supernatant (35 µl) was spotted onto phosphocellulose paper and then washed in 5 mM phosphoric acid and dried. The radioactivity on the phosphocellulose paper was quantified by liquid scintillation counting.
Detection of phosphoserine/threonine- and phosphotyrosine-containing proteins by western blotting
Following incubation (0, 30, 60, 90 and 120 min), 0.5 x 106 spermatozoa were washed (5 min, 600 g) to remove albumin and solubilized. Solubilization buffer [final concentration: 2% sodium dodecyl sulphate (SDS), 10% glycerol, 1.4% dithiothreitol (DTT), 62.5 mM Tris–HCl, pH 6.8, 0.1 mM sodium orthovanadate, 10 nM okadaic acid and 50 nM calyculin A] was added to each sample and boiled at 100°C for 5 min, sonicated and centrifuged at 14 000 g for 5 min. Proteins were separated by electrophoresis on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (10%) gels and electrotransferred onto nitrocellulose membrane. Nonspecific binding sites on membranes were blocked with either 5% BSA (w/v) or 5% (w/v) dry skimmed milk in Tris-buffered saline (0.9% NaCl, 20 mM Tris–HCl, pH 7.8) supplemented with 0.1% Tween-20 (TTBS) for the detection of phospho-serine/threonine and phosphotyrosine proteins, respectively. The membranes were incubated overnight at 4°C or 1 h at room temperature with the anti-phospho(serine/threonine) (1/1000) PKA substrate or anti-phosphotyrosine (1/10 000) antibodies, respectively. The membranes were then extensively washed with TTBS, incubated with corresponding secondary antibodies conjugated with horseradish peroxidase for 1 h and again extensively washed with TTBS. Positive immunoreactive bands were detected by chemiluminescence using LumiGLO (LumiGLO, Insight Biotechnology Ltd, Wembley, Middlesex, UK), an enhanced chemiluminescence kit, according to the manufacturer’s instructions. Silver staining of the proteins transferred on the nitrocellulose membrane was performed after the detection to confirm equal protein loading for all samples (Jacobson and Karsnas, 1990).
Detection of phosphotyrosine proteins by immunocytochemistry
Following incubation, 2 x 105 spermatozoa were smeared onto a slide, fixed in 100% methanol (30 min, –20°C) and washed in phosphate-buffered saline (PBS) (128 mM NaCl, 2 mM KCl, 8 mM Na2PO4, 2 mM KH2PO4 and 1 mM sodium azide, pH 7.5). The anti-phosphotyrosine antibody (1/500) was added to slides for 30 min. Slides were then washed in PBS and incubated with a fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse antibody for a further 30 min. Slides were mounted in fluorescence mounting medium. Two hundred cells were scored per slide.
Evaluation of sperm hyperactivation
Spermatozoa were incubated in IVF medium, CM or IVF sperm buffer. Samples were loaded into MicrocellTM-HAC chambers (50 µm depth) and motility assessed at T0, 30 and 90 min using computer-assisted semen analysis (CASA), Hamilton-Thorne IVOS version 10.9i. Percentage of progressive motility and hyperactivation were evaluated as defined previously (Mortimer, 2000). Motility data from at least 10 fields and 200 motile sperm were measured per treatment.
Assessment of progesterone-induced acrosome reaction
Spermatozoa were incubated in IVF medium, CM, IVF sperm buffer (SB) or NCM for 0, 30 and 90 min. After incubation, aliquots of spermatozoa were treated with 3.2 µM progesterone or dimethylsulphoxide (DMSO) as a control (0.05%) for 5 min as described previously (Kirkman-Brown et al., 2002). Propidium iodide (0.2 µg/ml) was used for viability determination. Cells were washed with PBS and air-dried onto slides (2 x 105 cells). Slides were subsequently permeabilized with methanol (2 min) and thoroughly washed in PBS. Cells were stained for acrosomal status using fluorescent Pisum sativum lectin (FITC-PSA) for 1 h at room temperature in a humid chamber. Slides were washed extensively with distilled water and a coverslip mounted with DakoCytomation fluorescence mounting medium. A total of 200 viable spermatozoa were scored. Acrosomal status was assessed as described by Mendoza et al. (1992): acrosome-intact cells showed a fluorescent acrosomal region and acrosome-reacted cells retained fluorescent labelling only in the equatorial region or occasionally retained no fluorescence at all. The percentage of spontaneously acrosome-reacted spermatozoa (DMSO treated cells) for each individual experiment was subtracted from the percentage of acrosome reacted cells obtained after progesterone treatment to give the percentage of cells which underwent acrosome reaction because of progesterone treatment.
Statistical analysis
Data were taken from at least three different donors and numerical data were averaged. Data were analysed with GraphPad PRISM® Version 3.03. Analysis of variance (ANOVA) (two-tailed, paired values) was used to test the differences in PKA activity, percentage of tyrosine phosphorylation on spermatozoa and progesterone-induced acrosome reaction. Hyperactivation levels within the same group at different time points during the experiment were compared using the Friedman test (nonparametric repeated measures test) to identify statistically significant differences. A difference was considered statistically significant at P < 0.05.
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Results |
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PKA activity and PKA-dependent protein phosphorylation in sperm incubated in different media
Levels of PKA activity in spermatozoa incubated in NCM remained low and constant throughout the 90-min incubation (Figure 1A). In contrast, spermatozoa incubated in media that support capacitation (IVF medium and CM) for less than 1 min (T0) showed an immediate greater than two-fold increase in PKA activity compared with cells suspended in NCM (Figure 1A). Levels dropped over the subsequent 15 min before reaching a plateau at 30 min. PKA activities of cells incubated in CM and IVF medium were statistically similar but were significantly different (at T0, 30 and 90 min) to those of spermatozoa incubated in NCM (Figure 1A). Maximum PKA activity measured using dbcAMP and IBMX in the assay mixture was similar in all three media (data not shown), indicating that the differences observed in Figure 1A reflect effects of the media on PKA activity.
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To measure PKA-dependent serine/threonine phosphorylation, we used an antibody that detects a phosphorylated motif, arginine (Arg)-X-X-Ser/Thr (X represents any amino acid), characteristic of PKA substrate (Bruce et al., 2002; Grøndborg et al., 2002). PKA-dependent serine/threonine phosphorylation in the different media showed levels of phosphorylation consistent with the observed differences in PKA activity. In spermatozoa suspended in NCM, levels of phosphorylation were low throughout the incubation period (except for constitutive phosphorylation of two bands at 
55 kDa; Figure 1B and C). However, in both capacitating media, levels of phosphorylation increased immediately (T0) to maximum intensity, levels in cells incubated in CM appearing to be slightly greater (Figure 1B and C). Phosphorylation was clearly lower after 120 min incubation (Figure 1B), a pattern similar to that observed by O’Flaherty et al. (2004). IVF sperm buffer, which contains 10 mM bicarbonate (compared to 25 mM and 0 mM in capacitating and NCM) showed an intermediate level of phosphorylation after 90 min of incubation (Figure 1C).
Protein tyrosine phosphorylation in sperm incubated in different media
We have investigated tyrosine phosphorylation of two major proteins of 105 and 81 kDa (p105/81) (Leclerc et al., 1996; Lefièvre et al., 2000; Kirkman-Brown et al., 2002). Similar to serine/threonine phosphorylation, levels of protein tyrosine phosphorylation were low in spermatozoa incubated under non-capacitating conditions (Figure 2A and B). However, when cells were incubated in either of the two capacitating media, levels of phosphorylation increased in a time-dependent manner, reaching a maximum sooner (60 min) in IVF medium than in CM (90 min; Figure 2A). Moreover, levels of tyrosine phosphorylation were higher in cells incubated in IVF media compared with CM throughout the 2-h incubation (Figure 2A and B). Incubation of spermatozoa in IVF sperm buffer induced intermediate levels of protein tyrosine phosphorylation (Figure 2B). Levels of tyrosine phosphorylation obtained at T0 were always significantly lower than those obtained after 60, 90 or 120 min incubation in the different media, but the intensity of phosphorylation varied from donor to donor (data not shown).
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Under our experimental conditions, spermatozoa displayed tyrosine phosphorylation only on the flagellum (Figure 2D). The proportion of cells in which phosphorylation was detectable depended on both the incubation medium and the duration of incubation. In cells incubated in IVF medium or CM there was a time-dependent increase, such that the proportion of labelled cells was significantly higher after 90 min than at T0 or 30 min. However, the proportion of labelled cells was significantly higher in IVF medium. In non-capacitating media and IVF sperm buffer, the percentage of cells with detectable tail phosphorylation remained low, even after 90 min (Figure 2C).
Effect of different media on sperm hyperactivation and progesterone-induced acrosome reaction
Levels of hyperactivation in spermatozoa incubated in either CM or IVF sperm buffer were constant and did not differ throughout the 90-min incubation (P = 1.00 and P = 0.94) (Figure 3A). Although not statistically significant, the percentage of spermatozoa displaying hyperactivation showed an apparent time dependent increase in spermatozoa incubated in IVF medium, P-value approaching the level of significance (P = 0.07, Figure 3A). The percentage of sperm showing progressive motility was also evaluated to ensure that incubation conditions were not detrimental to sperm viability. No differences between the different incubation media were observed (data not shown). Sperm hyperactivation could not be reliably estimated in non-capacitated spermatozoa because under these conditions a significant proportion of spermatozoa attached to the slide.
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The rate of spontaneous live acrosome reaction in DMSO controls ranged from 0.7 to 2.7% and showed no trend with respect to the duration of incubation. Stimulation with 3.2 µM progesterone immediately (T0) or 30 min after suspending spermatozoa in IVF medium, CM, IVF sperm buffer or NCM failed to cause a significant increase in the proportion of acrosome-reacted cells (Figure 3B). However, spermatozoa incubated for 90 min in IVF medium responded to progesterone stimulation with a significantly higher level of acrosome reacted cells than in parallel controls (DMSO). The response of cells incubated for 90 min in IVF medium was also significantly greater than that of cells incubated for 90 min in any of the other incubation media (Figure 3B), an observation consistent with the levels of tyrosine phosphorylation, after 90-min incubation in various media.
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Discussion |
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IVF is a well-established and successful technology but remarkably, as of yet, no studies have looked at the effect of a commercially available IVF media on markers of sperm capacitation or on associated physiological events such as sperm hyperactivation and progesterone-induced acrosome reaction. Our results demonstrate that IVF medium accelerates sperm capacitation compared with a standard laboratory CM. Tyrosine phosphorylation (Figure 2), incidence of hyperactivation (Figure 3A) and induction of acrosome reaction by progesterone (Figure 3B) were all clearly enhanced in IVF medium. Incubation in IVF sperm buffer (HEPES-buffered solution containing 10 mM bicarbonate), used for sperm preparation, increased serine/threonine and tyrosine phosphorylation over those of cells incubated in NCM but was less effective than IVF medium or CM (Figures 1 and 2). After 90-min incubation, hyperactivation and progesterone-induced acrosome reaction were not stimulated by IVF sperm buffer (Figure 3). The functional effect of this buffer on sperm capacitation has not been evaluated previously and our results suggest that spermatozoa require more than 10 mM bicarbonate to support sperm capacitation at least during short incubation.
Cyclic AMP is a central regulator of several key events of human sperm physiology, including motility, capacitation and acrosome reaction (De Jonge et al., 1991; Leclerc et al., 1996; Lefièvre et al., 2000). PKA, a cAMP-dependent kinase that phosphorylates proteins on serine/threonine residues (Arg-X-X-motif), has been implicated in tyrosine phosphorylation and associated stimulation of sperm motility (Leclerc et al., 1996; Aitken et al., 1998; Bajpai and Doncel, 2003; Luconi et al., 2005). Bicarbonate, present in capacitating media (25 mM in IVF medium and 26 mM in CM), plays a pivotal role in the stimulation of the soluble adenylyl cyclase (Chen et al., 2000), a key enzyme for the production of cAMP in sperm. The importance of bicarbonate in sperm capacitation is, however, best illustrated in Figure 1A. In the first minute of incubation (T0), IVF medium and CM both provoke an immediate greater than two-fold increase in PKA activity compared with NCM (without bicarbonate). This rapid rise correlates with the immediate increase in PKA-dependent serine-threonine phosphorylation of sperm proteins observed when spermatozoa were incubated under capacitating conditions (Figure 1B and C). Clearly, upon suspension of spermatozoa in capacitating media, PKA activation and PKA-dependent serine/threonine phosphorylation are very early events associated with sperm capacitation. It has been suggested recently that protein tyrosine kinase acts upstream of PKA activation; acting at the beginning of capacitation (O’Flaherty et al., 2004; Luconi et al., 2005) through a non-receptor type protein tyrosine kinase (O’Flaherty et al., 2004). Early tyrosine phosphorylation of AKAP3 (leading to increased binding to PKA) is believed to cause recruitment and activation of the enzyme leading to an increase in sperm motility (Luconi et al., 2005). If an initial wave of tyrosine phosphorylation contributes to PKA activation in our experiments it must be complete in <1 min or must be undetectable with our phosphotyrosine detection technique.
Interestingly, although CM and IVF medium were clearly different in their abilities to induce tyrosine phosphorylation and enhance functional characteristics associated with capacitation (Figures 2 and 3), PKA activity and serine/threonine phosphorylation were indistinguishable in these two capacitating media (Figure 1). Thus the stimulatory effect of IVF medium is apparently not an effect of enhanced activation of the cAMP signalling pathway. Consistent with this conclusion, H89, a widely used inhibitor of PKA activity, was shown to significantly inhibit PKA activity without affecting tyrosine kinase activity in capacitating sperm (Bajpai and Doncel, 2003). Furthermore we (unpublished data) and others (Bajpai and Doncel, 2003; Luconi et al., 2005) see little effect of H89 (10–50 µM) on sperm tyrosine phosphorylation of p105/81 proteins obtained under capacitating conditions. These observations suggest that activation of PKA may act as a ‘switch’ for stimulation of protein tyrosine phosphorylation associated with human sperm capacitation and motility. A threshold of PKA activity (which persists in the presence of H89) may be required for stimulation of tyrosine phosphorylation, but other factors (present in IVF medium) may determine the magnitude and rate of the effect. Alternatively, a second, PKA-independent pathway that is strongly activated in IVF medium, may regulate tyrosine kinase/phosphatases and downstream tyrosine phosphorylation. IVF medium contains several components that are absent in CM and which may contribute to stimulation of capacitation in this medium including glycine, taurine, glutamine and a selection of essential and nonessential amino acids as well as increased albumin levels and a different source of albumin (3% human serum albumin compared to 0.3% of bovine serum albumin in CM). Our observations strongly suggest that other factors contribute to the regulation of protein tyrosine kinase/phosphatases that occurs in cells incubated in IVF medium.
In summary, we have shown that after 90-min incubation in IVF sperm buffer (used for sperm preparation), sperm capacitation was not stimulated but that IVF medium accelerates capacitation (compared with a standard laboratory CM). The data strongly suggest that factors acting through signalling pathways other than bicarbonate/cAMP/PKA, may be responsible for this effect. Further studies should be performed to elucidate the biochemical changes that occur in spermatozoa during IVF as these may be clinically relevant. Finally, we propose that the IVF medium represents an excellent standardized medium for studying capacitation in human spermatozoa in vitro.
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Acknowledgements |
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We are grateful to our laboratory colleagues for assistance and encouragement during the course of this work. The authors thank Bayard Storey and Christopher De Jonge for critical reading of the paper. Thanks also to Aidan McMahon from Cook®, UK, for providing us with information about their Sydney IVF sperm buffer. This work was supported by the Birmingham Women’s Healthcare NHS Trust to C.L.R.B., the Lord Dowding Fund for Humane Research to C.L.R.B. and Fonds de recherche en santé du Québec to L.L.
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Submitted on April 8, 2005; accepted on May 11, 2005.
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